Contrasting patterns of Andean diversification among three diverse clades of Neotropical clearwing butterflies

The Neotropical region is the most biodiverse on Earth, in a large part due to the highly diverse tropical Andean biota. The Andes are a potentially important driver of diversification within the mountains and for neighboring regions. We compared the role of the Andes in diversification among three subtribes of Ithomiini butterflies endemic to the Neotropics, Dircennina, Oleriina, and Godyridina. The diversification patterns of Godyridina have been studied previously. Here, we generate the first time‐calibrated phylogeny for the largest ithomiine subtribe, Dircennina, and we reanalyze a published phylogeny of Oleriina to test different biogeographic scenarios involving the Andes within an identical framework. We found common diversification patterns across the three subtribes, as well as major differences. In Dircennina and Oleriina, our results reveal a congruent pattern of diversification related to the Andes with an Andean origin, which contrasts with the Amazonian origin and multiple Andean colonizations of Godyridina. In each of the three subtribes, a clade diversified in the Northern Andes at a faster rate. Diversification within Amazonia occurred in Oleriina and Godyridina, while virtually no speciation occurred in Dircennina in this region. Dircennina was therefore characterized by higher diversification rates within the Andes compared to non‐Andean regions, while in Oleriina and Godyridina, we found no difference between these regions. Our results and discussion highlight the importance of comparative approaches in biogeographic studies.     

Piezoelectric biosensors for biorecognition analysis: application to the kinetic study of HIV-1 Vif protein binding to recombinant antibodies

In this work three piezoelectric sensors modified with anti-HIV-1 Vif (virion infectivity factor) single fragment antibodies (4BL scFV), single domains (VH) and camelized single domains (VHD) were constructed and used to detect HIV1 Vif in liquid samples. Dithio-bis-succinimidyl-undecanoate (DSU) and 11-hydroxy-1-undecanethiol (HUT) mixed self assembled monolayers (SAM) were generated at the sensors surface onto which the antibodies were immobilized. All sensors detected specifically the target HIV1-Vif antigen in solution and no unspecific binding was monitored. Impedance analysis was performed to quantify electroacoustic and viscoelastic interferences during antibody immobilization and antigen recognition. The elimination of such interferences enabled the quantitative use of the piezoelectric immunosensors to estimate the antibody surface density as well as antigen binding and equilibrium constants. In spite of the possible limitation regarding mass transport and other related molecular phenomena, which were not considered in the binding model used, this work demonstrates the usefulness of piezoelectric biosensors in biorecognition analysis and evidences the advantages on using simultaneous impedance analysis to bring analytical significance to measured data, and thus to improve piezoelectric sensors sensitivity and applicability.     

Leishmanicidal effect of Spiranthera odoratíssima (Rutaceae) and its isolated alkaloid skimmianine occurs by a nitric oxide dependent mechanism

Leishmaniasis is one of the neglected diseases. High cost, systemic toxicity, and diminished efficacy due to development of resistance by the parasites has a negative impact on the current treatment options. Thus, the search for a new, effective and safer anti-leishmanial drug becomes of paramount importance. Compounds derived from natural products may be a better and cheaper source in this regard. This study evaluated the in vitro anti-leishmanial activity of Spiranthera odoratíssima (Rutaceae) fractions and isolated compounds, using promastigote and amastigote forms of different Leishmania species. J774 A.1 macrophage was used as the parasite host cell for the in vitro assays. Evaluations of cytoxicity, nitric oxide (NO), interleukin-10 and in silico analysis were carried out. In vitro experiments showed that the fruit hexanic fraction (Fhf) and its alkaloid skimmianine (Skm) have a significant (P<0·001) effect against L. braziliensis. This anti-L. braziliensis activity of Fhf and Skm was due to increased production of NO and attenuation of IL-10 production in the macrophages at concentrations ranging from 1·6 to 40·0 μg/ml. The in silico assay demonstrated significant interaction between Skm and amino acid residues of NOS2. Skm is thus a promising drug candidate for L. braziliensis due to its potent immunomodulatory activity.     

Mutations in methylenetetrahydrofolate reductase and in cysthationine beta synthase: is there a link to homocysteine levels in peripheral arterial disease?

Peripheral arterial disease (PAD) is an atherosclerotic disturbance characterized by a progressive obstruction of lower limb arteries. Many risk factors associated with PAD development have being reported in the literature. The present study aimed to investigate whether mutations in the methylenetetrahydrofolate reductase (MTHFR) or in the cystathionine beta synthase (CBS) genes are associated with higher levels of homocysteine and the risk of PAD in patients from Brazil. This study analyzed 39 patients with PAD and 32 without PAD in whom risk factors and C677T mutations in the MTHFR gene and both 844ins68 and T833C mutations in the CBS gene were investigated. Although higher levels of homocysteine could be observed in patients with PAD compared to controls, no association between the increase of homocysteine and the frequency of C677T, 844ins68, and T833C mutations could be observed. The results suggest that these mutations do not appear to be related to either homocysteine levels or the development of the disease. However, hyperhomocysteinemia and smoking are important factors in PAD development.     

Essential oils from Azorean Laurus azorica

The essential oils isolated from leaves of ten and from unripe berries of eight populations of Laurus azorica (Seub.) Franco, collected on five islands of the Azorean archipelago, were analysed by GC and GC-MS. All oil samples were dominated by their monoterpene fraction (60-94%), alpha-pinene (15-37%) and 1,8-cineole (12-31%) being the main components of the leaf oils, while trans-beta-ocimene (27-45%) and alpha-pinene (12-22%) were the main components of the oils from the berries. The sesquiterpene fractions of the oils ranged from 3 to 17% and the main components were beta-caryophyllene (traces-8%) and beta-elemene (traces-3%) both in the leaf and berry oils. Some phenylpropanoid components were also present, in total amounting to 17%, trans-cinnamyl acetate (215% of the leaf oils) being the main component of this fraction. Cluster analysis of the enantiomeric composition of alpha- and beta-pinene in the oils from the leaves clearly showed two groups, one constituted by the two populations growing on the island S. Jorge, and the other constituted by the remaining populations.     

From Mesolithic hunters to Iron Age herders: a unique record of woodland use from eastern central Europe (Czech Republic)

Vegetation History and Archaeobotany - In a continuous, perfectly stratified sedimentary sequence which was discovered under a large sandstone overhang in northern Bohemia, Czech Republic, we...     

Patterns of diversifying selection in the phytotoxin-like scr74 gene family of Phytophthora infestans

Phytophthora infestans, the organism responsible for the Irish famine, causes late blight, a re-emerging disease of potato and tomato. Little is known about the molecular evolution of P. infestans genes. To identify candidate effector genes (virulence or avirulence genes) that may have co-evolved with the host, we mined expressed sequence tag (EST) data from infection stages of P. infestans for secreted and potentially polymorphic genes. This led to the identification of scr74, a gene that encodes a predicted 74-amino acid secreted cysteine-rich protein with similarity to the Phytophthora cactorum phytotoxin PcF. The expression of scr74 was upregulated approximately 60-fold 2 to 4 days after inoculation of tomato and was also significantly induced during early stages of colonization of potato. The scr74 gene was found to belong to a highly polymorphic gene family within P. infestans with 21 different sequences identified. Using the approximate and maximum likelihood (ML) methods, we found that diversifying selection likely caused the extensive polymorphism observed within the scr74 gene family. Pairwise comparisons of 17 scr74 sequences revealed elevated ratios of nonsynonymous to synonymous nucleotide-substitution rates, particularly in the mature region of the proteins. Using ML, all 21 polymorphic amino acid sites were identified to be under diversifying selection. Of these 21 amino acids, 19 are located in the mature protein region, suggesting that selection may have acted on the functional portions of the proteins. Further investigation of gene copy number and organization revealed that the scr74 gene family comprises at least three copies located in a region of no more than 300 kb of the P. infestans genome. We found evidence that recombination contributed to sequence divergence within at least one gene locus. These results led us to propose an evolutionary model that involves gene duplication and recombination, followed by functional divergence of scr74 genes. This study provides support for using diversifying selection as a criterion for identifying candidate effector genes from sequence databases.     

High dietary salt decreases antioxidant defenses in the liver of fructose-fed insulin-resistant rats

In this study we investigated the hypothesis that a high-salt diet to hyperinsulinemic rats might impair antioxidant defense owing to its involvement in the activation of sodium reabsorption to lead to higher oxidative stress. Rats were fed a standard (CON), a high-salt (HS), or a high-fructose (HF) diet for 10 weeks after which, 50% of the animals belonging to the HF group were switched to a regimen of high-fructose and high-salt diet (HFS) for 10 more weeks, while the other groups were fed with their respective diets. Animals were then euthanized and their blood and liver were examined. Fasting plasma glucose was found to be significantly higher (approximately 50%) in fructose-fed rats than in the control and HS rats, whereas fat liver also differed in these animals, producing steatosis. Feeding fructose-fed rats with the high-salt diet triggered hyperinsulinemia and lowered insulin sensitivity, which led to increased levels of serum sodium compared to the HS group. This resulted in membrane perturbation, which in the presence of steatosis potentially enhanced hepatic lipid peroxidation, thereby decreasing the level of antioxidant defenses, as shown by GSH/GSSG ratio (HFS rats, 7.098±2.1 versus CON rats, 13.2±6.1) and superoxide dismutase (HFS rats, 2.1±0.05 versus CON rats, 2.3±0.1%), and catalase (HFS rats, 526.6±88.6 versus CON rats, 745.8±228.7 U/mg ptn) activities. Our results indicate that consumption of a salt-rich diet by insulin-resistant rats may lead to regulation of sodium reabsorption, worsening hepatic lipid peroxidation associated with impaired antioxidant defenses.     

Nation-wide litter fall data from 21 forests of the Monitoring Sites 1000 Project in Japan

This data paper reports litter fall data collected in a network of 21 forest sites in Japan. This is the largest litter fall data set freely available in Japan to date. The network is a part of the Monitoring Sites 1000 Project launched by the Ministry of the Environment, Japan. It covers subarctic to subtropical climate zones and the four major forest types in Japan. Twenty-three permanent plots in which usually 25 litter traps were installed were established in old-growth or secondary natural forests. Litter falls were collected monthly from 2004, and sorted into leaves, branches, reproductive structures and miscellaneous. The data provide seasonal patterns and inter-annual dynamics of litter falls, and their geographical patterns, and offer good opportunities for meta-analyses and comparative studies among forests.     

Efficient genome editing and gene knockout in Setaria viridis with CRISPR/Cas9 directed gene editing by the non-homologous end-joining pathway

The CRISPR/Cas9 system has been used for genome editing in several organisms, including higher plants. This system induces site-specific mutations in the genome based on the nucleotide sequence of engineered guide RNAs. The complex genomes of C4 grasses makes genome editing a challenge in key grass crops like maize (Zea mays), sorghum (Sorghum bicolor), Brachiaria spp., switchgrass (Panicum virgatum), and sugarcane (Saccharum spp.). Setaria viridis is a diploid C4 grass widely used as a model for these C4 crop plants. Here, an optimized CRISPR/Cas9 binary vector that exploits the non-homologous end joining (NHEJ) system was used to knockout a green fluorescent protein (gfp) transgene in S. viridis accession A10.1. Transformation of embryogenic callus by A. tumefaciens generated ten glufosinate-ammonium resistant transgenic events. In the T0 generation, 60% of the events were biallelic mutants in the gfp transgene with no detectable accumulation of GFP protein and without insertions or deletions in predicted off-target sites. The gfp mutations generated by CRISPR/Cas9 were stable and displayed Mendelian segregation in the T1 generation. Altogether, the system described here is a highly efficient genome editing system for S. viridis, an important model plant for functional genomics studies in C4 grasses. Also, this system is a potential tool for improvement of agronomic traits in C4 crop plants with complex genomes.